Valoración Geriátrica Integral

Antibodies (Ab) are soluble glycoproteins belonging to the immunoglobulin (Ig) family (Lòpez-Ribot et al., 2007; Schroeder et al , 2013). They are produced by plasma cells and they are found in the peripheral blood and external body fluids (Lòpez-Ribot et al., 2007). Antibodies have several functions: to neutralize viruses and identify and mark microbes and foreign/abnormal antigens for destruction (Lòpez-Ribot et al., 2007). Antibody-based therapy is now one of the most successful and important strategies for treating patients with many immune-mediated diseases and hematologic and solid tumors (Scott et al., 2012; Ecker et al., 2015).

1.4.1 Antibodies Structure

The antibodies structure consists of two identical copies of a heavy chain (HC) and two identical copies of a light chain (LC) in a Y-shaped structure. Each HC has approximately 50 kDa (450 amino acid residues) and each LC 25 kDa (212 amino acid residues) (Lòpez-Ribot et al., 2007; Schroeder et al, 2013). The heavy and light chains are linked by disulfide bonds (Williams et al, 1988; Schroeder et al, 2013). The N-terminal region of the Ab is characterized by sequence variability regions in both heavy (VH) and light (VL) chains (Lòpez-Ribot et al.,

The Ab also contains a hinge region between the CH1 and the CH2 that confers flexibility

to the two antigen-binding sites to operate independently, which is important due to the space between the protein molecules or the microbes (Lòpez-Ribot et al., 2007). The remaining Ab sequence is relatively constant either in the LC (CL) or in the HC (CH1, CH2, and CH3 regions).

Antibodies can also be divided into three fragments: two fragment antigen-binding (Fab) and a fraction crystallized (Fc). The Fab fragment is constituted by the VL, CL, VH, and CH1 regions,

and is responsible for the binding to the antigen. The Fc fragment contains the CH2 and CH3 regions and provides a binding site to immunocompetent cells receptors (Lòpez-Ribot et al., 2007).

There are five different Ig classes (isotypes) of Ab molecules based on the number of Y units and the structure of its carboxy (C)-terminal part on the HC. The five classes of Igs are: IgG, IgM, IgA, IgE and IgD. They differ in their biological properties, functional locations and ability to react with different antigens (Goldsby et al., 2003; Murphy, 2012).

There are two types of LC, the lambda (λ) and kappa (κ), based on small differences in the polypetide sequence. However, each class can have either a λ or κ LC and no functional differences have been found between them (Schroeder et al, 2013).

The Ab binding capacity is influenced by its affinity and avidity to the antigen. Affinity is the strength of the interaction between the Ab and the antigen. Therefore, high affinity means stronger connections to the antigen at a lower Ab concentration. Avidity is the combined strength of multiple interactions between the Ab and the antigen epitopes. It is dependent on the number of epitopes and number of antibody-combining sites (Lòpez-Ribot et al., 2007).

Figure 1.12. Schematic diagram of Ig structure. The Ab is constituted by two light chains and two heavy

chains each organized in variable regions (VH, VL) and constant regions (CH1, CH2, CH3, CL) linked together by disulfide bonds. In the centre of the molecule there is the hinge region, represented here as “papain cleavage site”. Papain is an enzyme that digests the molecule and divides it into three - two Fab fragments and one Fc fragment. Rose and red – light chains and Blue – heavy chains (Legninger, 5th edition).

The Ab specificity is determined by the variable regions (VL and VH). These regions

contain amino acid sequences that create a three-dimensional structure specific for each antigen. These sequences are called complementary-determining regions (CDRs), and each chain contains three CDRs (CDR1, CDR2, CDR3) (Lòpez-Ribot et al., 2007). The CDRs in the HC correspond to the amino acid residues 30 to 36, 49 to 65 and 95 to 103, while in the LC correspond to the amino acids 28 to 35, 49 to 59 and 92 to 103 (Murphy, 2011). The rest of the variable domain has a more constant sequence termed the framework regions (FR). There are four frameworks, FR1, FR2, FR3, and FR4, responsible for the CDRs positioning on the surface of the chain creating a hypervariable region at the end of each Ab (Murphy, 2011).CDRs amino acid sequences determine the shape and ionic properties of the antigen-binding site, determining the specificity of the Ab (Murphy, 2011).

The Ab diversity is generated by somatic recombination in B lymphocytes, where the variable gene segments are linked to other gene segments. LC contains VL, JL, and CL gene segments and the HC contains VH, DH, JH, and CH gene segments (Figure 1.13). CL gene segments encode the constant regions (Goldsby et al, 2003). In the assembled LC variable domain, the VL gene segment encodes FR1 to 3, CDR1 and 2, and two thirds of CDR3, while JL encodes the rest of CDR3 and FR4. In the HC variable domain, VH gene segment encodes FR1 to 3, CDR1 and 2 and JH encode FR4. The CDR3 is generated in developing B cells by the joining process, containing the entire DH as well as portions of VH and JH gene segments (Paul, 2013).

Figure 1.13. Representation of the chromosomal organization of the Ig heavy and light gene clusters. Genes of the heavy chain variable region are divided into the VH, DH and JH gene regions. The

genes in the light chain variable region are divided into the VL and the JL gene regions (adapted from https://www.kyowakirin.com/antibody/basics/diversity.html).

Being the Ab glycoproteins, they suffer glycosylation as modifications. Glycosylation alters the charge profile of the Ab and can affect its stability and potency (Mo et al., 2018). Usually, an Ab is glycosylated in the Fc domain with an N-linked glycosylation on Asn297 on each of the two CH2 domains that have been shown to have little impact on Fc functions.

However, the CDRs are unique to each Ab and glycosylation in the CDRs may affect antigen binding (Mo et al., 2018). Variation in glycosylation is observed between Ab molecules as well as within the two chains on the same molecule due to differences in terminal sialic acid, galactose, N-acetyl glucosamine, and fucosylation of the core. The glycans on the CH2 interact with a hydrophobic pocket on the Fc domain that stabilizes the Ab structure (Liu et al., 2006; Sibéril et al., 2006; Schroeder et al, 2013).

1.4.2 Monoclonal Antibodies

Monoclonal antibodies (mAb) are monospecific Ab produced from identical immune cells that derive from a single clone, and are specific for a unique epitope (Xin et al, 2013). mAbs are now being used to treat many diseases, such as cancer, infectious diseases, allergy, asthma, and some autoimmune diseases, where they can neutralize substances, block receptors, bind to cells and modulate the host immune system (Schirrmann et al, 2011). Trastuzumab and pertuzumab are two examples of therapeutic mAbs used for the treatment of patients with HER2-overexpressing metastatic BC (Gianni et al., 2012; Lamy et al., 2017). The use of mAbs as therapeutic agents against Notch in recent years resulted in anti-tumor activity in different types of cancer, with the advantage of overcoming the gastrointestinal toxicities associated with GSIs (Wu et al., 2010; Sharma et al., 2012, 2013; Lamy et al., 2017). Different therapeutic approaches have been followed to develop mAbs towards various Notch signaling proteins. Some consists in targeting the receptor-ligand binding domain while others in blocking the intracellular Notch cleavage by γ-secretase enzyme through specific binding to the NRR (Wu et al., 2010; Sharma et al., 2012, 2013; Lamy et al., 2017). OncoMed Pharmaceuticals developed two therapeutic mAbs: bronticuzumab, targeting Notch1 receptor (Davis et al., 2013) and tarextumab, targeting both Notch2 and 3 receptors (Yen et al., 2015), to increase the efficacy of conventional BC therapies (Lamy et al., 2017). Bronticuzumab showed clinical benefits in phase I studies in patients with advanced solid tumors, in a patient with refractory HER2-negative BC and reduced the number of circulating tumor cells in a patient with colorectal cancer (Davis et al., 2013; Lamy et al., 2017). Tarextumab showed in a phase I study in patients with advanced solid tumors, to prolong stable disease in TNBC, sarcomas, and rectal cancer (Davis et al., 2013; Lamy et al., 2017). In both studies, small toxicity levels were obtained.

1.4.2.1

Antibody Formats

The antigen-binding fragments can be produced by biochemical digestion and include Fab, (Fab')2, and FV. The Fab fragment is composed of the LC and the variable domain and

CH1 from the HC, thus containing one antigen-binding site (Frenzel et al., 2013). Divalent (Fab')2 fragments have two antigen-binding regions, linked by disulfide bonds, and are produced by pepsin digestion of IgG or IgM Abs, which retains a portion of the hinge region. The Fv fragment is the smallest antigen-binding fragment and consists of one variable domain of the HC. The Fv fragments have low stability so single-chain Fv fragments (scFv) were developed, in which one variable region of each light and heavy chain are tethered together by a soluble linker, for stabilization of the molecule. scFvs are produced using genetic engineering

methods. scFv fragments have a complete binding site of an Ab and they are presently the most popular format, as the Fab, of recombinant Abs with wide application in healthcare and biotechnology (Frenzel et al., 2013). Evolution of this platform has also resulted in bi-scFvs, using a linker with 3-11 residues long, and in bispecific antibodies that can cross-link different antigens and thus hold promise as anti-cancer drugs. In addition to conventional Abs, camelid species contain a subset of HC Abs (hcAb) exclusively composed by HC homodimers lacking light chains. The Fab portions of these antibodies, called VHH, are the smallest antigen-binding

regions naturally found. They are stable and can be easily produced in huge quantity by using common simple protein expression systems (Muyldermans, 2013).

Figure 1.14. Different Ab formats. Representation of several Ab formats, including the intact IgG Ab

alongside various representations of Ab fragments. Image from https://www.antibodies- online.com/resources/18/1502/antibody-and-immunoglobulin-alternatives-part-1/).